Interlaboratory collaborative study on a flow cytometry method for lactic acid bacteria quantification in starter cultures, probiotics and fermented milk products according to ISO 193441 IDF 232

摘要

An international collaborative study of ISO 19344 IDF 232, Milk and milk products - Starter cultures, probiotics and fermented products - Quantification of lactic acid bacteria by flow cytometry was conducted to determine precision figures for the quantification of lactic acid bacteria and probiotic strains by flow cytometry in starter cultures, probiotics and fermented milk products. Ten sample types (two batches per sample type), obtained from representative commercially available matrices, wereprepared and dispatched to 15 participating laboratories located in five different countries. Laboratories analyzed the randomly coded samples in duplicate using the three staining protocols included in the international standard. After collection and preparation of the resulting data (both active and total fluorescent units per gram; AFU/g and TFU/g), statistical analyses were performed on the log10 transformed data in accordance with ISO 5725-2 and IDF requirements, and in close cooperation with the IDF Project Group on Statistics of Analytical Data - Interlaboratory Study Results. Four of the 900 potential original data for AFU and 34 of the 900 for TFU were missing. From the reported data for both AFU and TFU, 3.7% were identified as outliers and were removed prior to final calculation of precision figures. Most of the missing TFU data indicated the challenges associated with gating TFU in yoghurt samples as a result of the high concentration of particulate matter, despite careful sample preparation. Except for this problem, all laboratories were able to deliver acceptable datasets and none of the samples were over- or underestimated by more than one laboratory. Except for yoghurts, the repeatability limits (r) and reproducibility limits (/?) were calculated to be 0.06 and 0.45 for log10 AFU per gram and 0.07 and 0.38 for log10 TFU per gram. The corresponding values for yoghurts were 0.08 and 0.76 for log10 AFU per gram and 0.10 and 1.17 for log10 TFU per gram. The latter reproducibility value illustrates the challenges encountered when analyzing TFU in yoghurts. Furthermore, the equivalence of AFU results for the three different staining protocols was evaluated and demonstrated for most of the strains included in this study.